The present invention relates to a linker nucleoside, its preparation and use for the covalent bonding of biomolecules to oligonucleotides, in particular p-RNA oligonucleotides.
Pyranosylnucleic acids (p-NAs) are in general structures isomeric to the natural RNA, in which the pentose units are present in the pyranose form and are repetitively linked between the positions C-2xe2x80x2 and C-4xe2x80x2 by phosphodiester groups (FIG. 1). xe2x80x9cNucleobasexe2x80x9d is understood here as meaning the canonical nucleobases A, T, U, C, G, but also the pairs isoguanine/isocytosine and 2,6-diaminopurine/xanthine and, within the meaning of the present invention, also other purines and pyrimidines. p-NAs, namely the p-RNAs derived from ribose, were described for the first time by Eschenmoser et al. (Pitsch, S. et al. Helv. Chim. Acta 1993, 76, 2161; Pitsch, S. et al. Helv. Chim Acta 1995, 78, 1621; Angew. Chem. 1996, 108, 1619-1623). They exclusively form so-called Watson-Crick-paired, i.e. purine-pyrimidine- and purine-purine-paired, antiparallel, reversibly xe2x80x9cmeltingxe2x80x9d, quasi-linear and stable duplexes. Homochiral p-RNA strands of the opposite sense of chirality likewise pair controllably and are strictly non-helical in the duplex formed. This specificity, which is valuable for the synthesis of supramolecular units, is connected with the relatively low flexibility of the ribopyranose phosphate backbone and also with the strong inclination of the base plane to the strand axis and the tendency resulting from this for intercatenary base stacking in the resulting duplex and can finally be attributed to the participation of a 2xe2x80x2,4xe2x80x2-cis-disubstituted ribopyranose ring in the synthesis of the backbone. These significantly better pairing properties make p-NAs preferred pairing systems, compared with DNA and RNA, for use in the synthesis of supramolecular units. They form a pairing system which is orthogonal to natural nucleic acids, i.e. they do not pair with DNAs and RNAs occurring in the natural form, which is particularly of importance in the diagnostic field.
Eschenmoser et al. (1993, supra) has for the first time prepared a p-RNA, as shown in FIG. 2 and explained below.
In this connection, a suitable protected nucleobase was reacted with the anomer mixture of the tetrabenzoylribopyranose by action of bis(trimethylsilyl)acetamide and a Lewis acid such as, for example, trimethylsilyl trifluormethanesulphonate (analogously to H. Vorbrxc3xcggen, K. Krolikiewicz, B. Bennua, Chem. Ber. 1981, 114, 1234.). Under the action of base (NaOH in THF/methanol/water in the case of the purines; saturated ammonia in MeOH in the case of the pyrimidines), the acyl protective groups were removed from the sugar, and the product was protected in the 3xe2x80x2,4xe2x80x2-position under acidic catalysis with p-anisaldehyde dimethyl acetal. The diastereomer mixture was isolated in the 2xe2x80x2-position, the 3xe2x80x2, 4xe2x80x2-methoxybenzylidene-protected 2xe2x80x2-benzoate was deacetalized by acidic treatment, e.g. with trifluoroacetic acid in methanol, and reacted with dimethoxytrityl chloride. The 2xe2x80x2xe2x86x923xe2x80x2 migration of the benzoate was initiated by treatment with p-nitrophenol/4-(dimethylamino)pyridine/triethylamine/pyridine/n-propanol. Almost all reactions were worked up by column chromatography. The key structural unit synthesized in this way, the 4xe2x80x2-DMT-3xe2x80x2-benzoyl-1xe2x80x2-nucleobase derivative of the ribopyranose, was then partly phosphitylated or bonded to a solid phase via a linker.
In the subsequent automated oligonucleotide synthesis, the carrier-bonded component in the 4xe2x80x2-position was repeatedly acidically deprotected, a phosphoramidite was coupled on under the action of a coupling reagent, e.g. a tetrazole derivative, still-free 4xe2x80x2-oxygen atoms were acetylated and the phosphorus atom was oxidized in order thus to obtain the oligomeric product. The remaining protective groups were then removed, and the product was purified and desalted by means of HPLC.
The disadvantage of the already known p-RNA oligonucleotides is that no methods are known to covalently bond other biomolecules to these oligonucleotides.
It was therefore the object of the present invention to make available suitable constructs which make possible covalent bonding of other biomolecules to oligonucleotides, in particular to p-RNA oligonucleotides.
A subject of the present invention is therefore a linker nucleoside of the formula (I) or (II), 
in which R1 is equal to H, OH, phosphoramidite, Hal where Hal is preferably equal to Br or Cl,
R2, R3 and R4 independently of one another, identically or differently, in each case are OCnH2nxe2x88x921 for formula (I) or (CnH2n)NR10R11 for formula (I) and (II) where
R10R11 is linked via a radical of the formula 
in which R12, R13, R14 and R15 independently of one another, identically or differently, in each case are H, CnH2n+1 or CnH2nxe2x88x921 or OR7, where R7 is equal to H, CnH2nxe2x88x921 or CnH2nxe2x88x921, xe2x80x94C(O)R where R8 is equal to a linear or branched, optionally substituted alkyl or aryl radical, preferably a phenyl radical, where n is equal to an integer from 1-12, preferably 1-8, in particular 1-4, X, Y and Z independently of one another, identically or differently, in each case are xe2x95x90Nxe2x80x94, xe2x95x90C(R9)xe2x80x94 or xe2x80x94N(R9)xe2x80x94 where R9 and R9xe2x80x2 independently of one another, identically or differently, in each case are H or CnH2n+1 or (CnH2n)NR10R11 having the abovementioned meanings, in a particular embodiment the radicals R2, R3 and R4 and the atoms X, Y and Z taken together have the meaning assigned to them by the structure of the linker nucleoside of the formula (I) or (II) as a pentopyranosyl- or pentofuranosylpurine, -2,6-diaminopurine, -6-purinthiol, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine, -xanthine, -hypoxanthine, -indole, -tryptamine, -N-phtaloyl-tryptamine, -caffeine, -theobromine, -theophylline or benzotriazole, and Sc1 and Sc2 independently of one another, identically or differently, in each case are H or a protective group selected from an acyl, trityl or allyloxycarbonyl group, preferably a benzoyl or 4, 4xe2x80x2-dimethoxytrityl (DMT) group, or a phosphoester(III), phosphoester(V), thiophosphate(V), phosphonate or phosphoramidite,
or of the formula (III) or (IV) 
in which R1xe2x80x2 is equal to H, OH, phosphoramidite or Hal where Hal is preferably equal to Br or Cl, R2xe2x80x2,R3xe2x80x2 and R4xe2x80x2 independently of one another, identically or differently, in each case for formula (III) OCnH2nxe2x88x921 where n is equal to an integer from 1-12, preferably 1-8, in particular 1-4, or for formula (III) and (IV) (CnH2n)NR10xe2x80x2R11xe2x80x2, where R11xe2x80x2, independently of one another has the abovementioned meaning of R10 or R11, and Xxe2x80x2 in each case is xe2x95x90Nxe2x80x94, xe2x95x90C(R9xe2x80x2)xe2x80x94 or xe2x80x94N(R9xe2x80x3)xe2x80x94, where R9xe2x80x2 and R9xe2x80x3 independently of one another have the above-mentioned meaning of R9 and R9xe2x80x2, in a particular embodiment the radicals R2xe2x80x2, R3xe2x80x2 and R4xe2x80x2 and the atom X taken together have the meaning assigned to it by the structure of the linker nucleoside of the formula (III) or (IV) as a pentopyranosyl- or pentofuranosylpyridine, -pyrimidine, -thymidine, -cytosine, -isocytosine, -uracil, and Sc1xe2x80x2 and Sc2xe2x80x2 have the abovementioned meaning of Sc1 and Sc2.
The pentose according to the invention is in general a ribose, arabinose, lyxose and/or xylose, preferably a ribopyranose, where the pentopyranosyl moiety can have the D configuration, but also the L configuration.
Customarily, the linker nucleoside according to the invention is a pentopyranosyl- or pentofuranosylpurine, -2,6-diaminopurine, -6-purinthiol, -pyridine, -pyrimidine, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine, -xanthine, -hypoxanthine, -thymidine, -cytosine, -isocytosine, -indole, -tryptamine, -N-phthaloyltryptamine, -uracil, -caffeine, -theobromine, -theophylline, -benzotriazol or -acridine, in particular a pentopyranosylpurine, -pyrimidine, -adenosine, -guanosine, -thymidine, -cytosine, -tryptamine, -N-phthalotryptamine or -uracil.
The linker nucleosides according to the invention are consequently compounds having functional groups which can covalently bond biomolecules, to, for example, nucleic acids occurring in their natural form or modified nucleic acids, such as DNA, RNA but also p-NAs, preferably pRNAs. For p-NAs, this is particularly advantageous, as in this case no linkers are yet known.
For example, among these are pentopyranosylnucleosides in which R2, R3, R4, R2xe2x80x2, R3xe2x80x2 and/or R4xe2x80x2 for formula (I), (II), (III) and (IV) is a 2-phthalimidoethyl or, for formula (I) and (Im), an allyloxy radical. Preferred uracil-based linkers according to the present invention are, for example, those in which the 5-position of the uracil has preferably been modified, e.g. N-phthaloylaminoethyluracil, but also indole-based linkers, preferably tryptamine derivatives, such as, for example, N-phthaloyltryptamine.
In a particular embodiment, for example, a linker according to formula (III) or (IV), in which R4xe2x80x2 is (CnH2n)NR10xe2x80x2R11xe2x80x2 and R10xe2x80x2R11xe2x80x2 is linked to the meaning already designated via a radical of the formula (V), is advantageously prepared by the following process:
(a) a compound of the formula (III) or (IV) where R4xe2x80x2 is equal to (CnH2n)OSc3 or (nH2n)Hal, in which n has the abovementioned meaning, Sc3 is a protective group, preferably a mesylate group, and Hal is chlorine or bromine, is reacted with an azide, preferably in DMF, then
(b) the reaction product from (a) is reduced, preferably using triphenylphosphine e.g. in pyridine, then
(c) the reaction product from (b) is reacted with an appropriate phthalimide, e. g. N-ethoxycarbonylphthalimide, and
(d) the reaction product from (c) is reacted with an appropriate protected pentose, e.g. ribose tetrabenzoate, and finally
(e) the protective groups are optionally removed, e.g. using methylate, and the product is then optionally converted into a phosphorylated unit which is suitable for oligonucleotide synthesis.
In addition, indole derivatives as linkers have the advantage of the ability to fluoresce and are therefore particularly preferred for nanotechnology applications which may concern the detection of very small amounts of substance. Thus indole-1-ribosides have already been described in N. N. Suvorov et al., Biol. Aktivn. Soedin., Akad. Nauk SSSR 1965, 60 and Tetrahedron 1967, 23, 4653. However, there is no analogous process for preparing 3-substituted derivates. In general, they are prepared via the formation of an aminal of the unprotected sugar component and an indoline, which is then converted into the indole-1-riboside by oxidation. The indole-1-glucoside and -1-arabinoside, for example, whose 3-substituted derivates were usually prepared by means of Vielsmeier reaction, have been described (Y. V. Dobriynin et al, Khim.-Farm. Zh. 1978, 12, 33). This way of introducing aminoethyl units into the 3-position of the indole is too complicated, however, for industrial application.
In a further particular embodiment, a linker according to formula (I) or (II), in which X and Y independently of one another, identically or differently, in each case are xe2x95x90C(R16) where R16 is equal to H or CnH2n and Z is xe2x95x90C(R16)xe2x80x94 where R16 is equal to (CnH2n)NR10R11 is therefore advantageously prepared, for example, by the following process:
(a) the corresponding indoline, e.g. N-phthaloyltryptamine, is reacted with a pentose, e.g. D-ribopyranose, to give the nucleoside triol, then
(b) the hydroxyl groups of the pentose moiety of the product from (a) are preferably protected with acyl groups, e.g. by means of acetic anhydride, then
(c) the product from (b) is oxidized, e.g. by means of 2,3-dichloro-5,6-dicyanoparaquinone, and
(d) if appropriate, the hydroxyl protective groups of the pentose moiety of the product from (c) are removed, for example by means of methylate, and then optionally converted into a phosphorylated unit which is suitable for oligonucleotide synthesis.
The processes described, however, cannot only be used in the case of ribopyranose, but also in the case of ribofuranose and 2xe2x80x2-deoxyribofuranoses or 2xe2x80x2-deoxyribopyranoses, which is particularly advantageous. As a nucleosidation partner of the sugars, tryptamine, in particular N-acyl derivates of tryptamine, especially N-phthaloyltryptamine, is preferably used. The remaining linker nucleosides can be prepared in an analogous manner or a manner known to the person skilled in the art.
In a further embodiment, the 4xe2x80x2-protected, preferably, the 3xe2x80x2, 4xe2x80x2-protected linker nucleosides are phosphitylated in a further step or bonded to a solid phase.
The phosphitylation is effected, for example, by means of monoallyl N-diisopropylchlorophosphoramidite in the presence of a base, e.g. N-ethyldiisopropylamine. The bonding of a protected pentopyranosylnucleoside according to the invention to a solid phase, e.g. xe2x80x9clong-chain-alkylamino-controlled pore glassxe2x80x9d (CPG, Sigma Chemie, Munich) can be carried out, for example, as described in Eschenmoser et al. (1993).
The compounds obtained serve, for example, for the preparation of pentopyranosylnucleic acids, which contain one of the linkers according to the invention.
A further subject of the present invention is therefore a process for the preparation of a nucleic acid, having the following steps:
(a) in a first step a protected nucleoside or a protected linker nucleoside is bonded to a solid phase and
(b) in a second step the 3xe2x80x2-, 4xe2x80x2-protected nucleoside bonded to a solid phase according to step (a) is lengthened by a phosphitylated 3xe2x80x2-, 4xe2x80x2-protected nucleoside or linker nucleoside, then oxidized, for example, by an aqueous iodine solution, and
(c) step (b) is repeated using identical or different nucleosides or linker nucleosides until the desired nucleic acid is present, the nucleic acid containing at least one inventive linker nucleoside.
A suitable coupling reagent is particularly benzimidazolium triflate, preferably after recrystallizing in acetonitrile and after dissolving in acetonitrile, as in contrast to 5-(4-nitrophenyl)-1H-tetrazole as a coupling reagent no blockage of the coupling reagent lines and contamination of the product takes place.
Furthermore, it is advantageous by means of addition of a salt, such as sodium chloride, to the protective-group-removing hydrazinolysis of oligonucleotides, in particular of p-NAs, preferably of p-RNAs, to protect pyrimidine bases, especially uracil and thymine, from a ring-opening which would destroy the oligonucleotide. Allyoxy groups can preferably be removed by palladium [Pd(O)] complexes e.g. before hydrazinolysis.
In a further particular embodiment, pentofuranosyl nucleosides, e.g. adenosine, guanosine, cytidine, thymidine and/or uracil occurring in their natural form, in addition to pentopyranosylnucleosides, can also be incorporated in step (a) and/or step (b), which leads, for example, to a mixed p-NA-DNA or p-NA-RNA.
In another particular embodiment, in a further step an allyloxy linker of the formula
Sc4NH(CnH2n)CH(OPSc5Sc6)CnH2n OSc7xe2x80x83xe2x80x83(VI),
in which Sc4 and Sc7 independently of one another, identically or differently, are in each case a protective group in particular selected from Fmoc and/or DMT, Sc5 and Sc6 independently of one another, identically or differently, are in each case an allyloxy and/or diisopropylamino group, can be incorporated. n has the meaning already mentioned above.
The present invention therefore also extends to an allyloxy linker of the formula
Sc4NH(CnH2n)CH(OPSc5Sc6)CnH2nSc7xe2x80x83xe2x80x83(VI),
in which Sc4 and Sc7 independently of one another, identically of differently, are in each case a protective group in particular selected from Fmoc and/or DMT, Sc5 and Sc6 independently of one another, identically or differently, are in each case an allyloxy and/or diisopropylamino group and n is as designated above.
A particularly preferred allyloxy linker is (2-(S)-N-Fmoc-O1-DMT-O2-allyloxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol).
Starting from, for example, lysine, it is thus possible in a few reaction steps to synthesize amino-terminal linkers which carry both an activatable phosphorus compound and an acid-labile protective group, such as DMT, and can therefore be easily used in automatable oligonucleotide synthesis (see, for example, P. S. Nelson et al., Nucleic Acid Res. 1989, 17, 7179; L. J. Arnold et al., WO 8902439). The repertoire was extended in the present invention by means of a lysine-based linker in which, instead of the otherwise customary cyanoethyl group on the phosphorus atom, an allyloxy group has been incorporated, and which can therefore be employed advantageously in the Noyori oligonucleotide method (R. Noyori, J. Am. Chem. Soc. 1990, 112, 1691-6).
In another particular embodiment, in a further step a lysine linker of the formula 
can be incorporated.
The present invention therefore also extends to a lysine linker of the formula 
A further subject of the present invention is therefore also a nucleic acid which contains at least one linker nucleoside according to the invention and optionally at least one allyloxy linker according to the invention. A pentopyranosylnucleic acid is particularly preferred, as p-NAs and in particular the p-RNAs form stable duplexes with one another and in general do not pair with DNAs and RNAs occurring in their natural form. This property makes p-NAs preferred pairing systems.
Such pairing systems are supramolecular systems of non-covalent interaction, which are distinguished by selectivity, stability and reversibility, and whose properties are preferably influenced thermodynamically, i.e. by temperature, pH and concentration. Such pairing systems can also be used, for example, on account of their selective properties as a xe2x80x9cmolecular adhesivexe2x80x9d for the bringing together of different metal clusters to give cluster associates having potentially novel properties [see, for example, R. L. Letsinger, et al., Nature 1996, 382, 607-9; P. G. Schultz et al., Nature 1996, 382, 609-11]. Consequently, the p-NAs are also suitable for use in the field of nanotechnology, for example for the production of novel materials, diagnostics and therapeutics and also microelectronic, photonic and optoelectronic components and for the controlled bringing together of molecular species to give supramolecular units, such as, for example, for the (combinatorial) synthesis of protein assemblies [see, for example, A. Lombardi, J. W. Bryson, W. F. DeGrado, Biopolymers (Pept. Sci.) 1997, 40, 495-504], as p-NAs form pairing systems which are strongly and thermodynamically controllable. A further application therefore especially arises in the diagnostic and drug discovery field due to the possibility of providing functional, preferably biological, units such as proteins or DNA/RNA sections, with a p-NA code which does not interfere with the natural nucleic acids (see, for example, WO93120242).
A further subject of the present invention is a conjugate comprising a linker nucleoside according to the invention or an inventive nucleic acid and a biomolecule.
Biomolecule is understood according to the present invention as antibody or functional moieties thereof or an enzyme, and also a nucleic acid such as DNA or RNA, or cell constituents such as lipids, glycoproteins, filaments constituents, or viruses, virus constituents such as capsids, viroids, and their derivatives such as, for example, acetates. Functional moieties of antibodies are, for example, Fv fragments (Skerra and Plxc3xcckthun (1988) Science 240, 1038), single-chain Fv fragments (scFv; Bird et al (1988), Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 5879) or Fab fragments (Better et al. (1988) Science 240, 1041).
The conjugates according to the invention of effector molecules and preferably peptide, but in contrast to PNA, selective and controllable pairing systems are advantageous if reversibly supramolecular assemblies are to be synthesized, whose action, such as, for example, binding, inhibition, induction of a physiological effect, differs from the action of the individual effector molecules.
An attempt to use peptide xe2x80x9cadhesivexe2x80x9d units for the formation of homo- or heterodimeric assemblies is described, for example, in WO 94/28173:
Association peptides (hexa- or heptapeptides) of a fixed sequence bring together effector units, such as, for example, proteins, to give supramolecular units. Such a method can maintain higher flexibility by means of controllability of this association process, which in general cannot be realized with the association peptides, but advantageously with the pairing systems of the present invention.
Thus, for example, WO 96/13613 describes a method for finding a substance which induces a biological action due to the multimerization of proteins by first determining a substance I by means of a test, which substance binds to a protein, then determining a second substance II which binds to a second protein and then linking the two substances I and II covalently via a linker such that dimerization of the two proteins is induced. This dimerization then brings about the desired biological effect. Such a method can maintain greater flexibility if the linking of the two substances I and II does not take place covalently, but by means of a pairing system such as the oligomer or conjugate according to the invention. As a result of the controllability of this pairing, for example by means of temperature or pH, the dimerization process of the proteins can be observed or its extent can be controlled. The pairing systems according to the invention have the advantage, for example, compared with the systems from WO 96/13522, that they are nuclease-stable.
A biomolecule, e.g. DNA or RNA, can be used for non-covalent linking to another biomolecule, e.g. DNA or RNA if both biomolecules contain sections which can bind to one another by formation of hydrogen bridges as a result of complementary sequences of nucleobases. Biomolecules of this type are found, for example, in analytical systems for signal amplification units, where a DNA molecule whose sequence is to be analyzed is immobilized on a solid carrier by means of such a non-covalent DNA linker on the one hand, and on the other hand is to be bonded (see, for example, S. Urdea, Bio/Technol. 1994, 12, 926 or U.S. Pat. No. 5,624,802) to a signal-amplifying branched DNA molecule (bDNA). A significant disadvantage of the last-described systems is that they are subject to date to the process for nucleic acid diagnosis by means of polymerase chain reaction (PCR) (K. Mullis, Methods Enzymol. 1987, 155, 335) with respect to sensitivity. This is to be attributed, inter alia, to the fact that the non-covalent bonding of the solid carrier to the DNA molecule to be analyzed does not always take place specifically, just like the non-covalent bonding of the DNA molecule to be analyzed, as a result of which a mixing of the functions xe2x80x9csequence recognitionxe2x80x9d and xe2x80x9cnon-covalent bondingxe2x80x9d occurs. The use of p-NAs as an orthogonal pairing system which does not intervene in the DNA or RNA pairing processes solves this problem advantageously, as a result of which the sensitivity of the analytical processes described can be markedly increased.
In a preferred embodiment, what is concerned here are p-RNA/DNA or p-RNA/RNA conjugates.
Conjugates are preferably used if the functions xe2x80x9csequence recognitionxe2x80x9d and xe2x80x9cnon-covalent bondingxe2x80x9d have to be realized in one molecule, as the conjugates according to the invention contain two pairing systems which are orthogonal to one another.
Both sequential and convergent processes are suitable for the preparation of conjugates.
In a sequential process, for example, after automated synthesis of a p-RNA oligomer has taken place directly on the same synthesizerxe2x80x94after readjustment of the reagents and of the coupling protocolxe2x80x94e.g. a DNA oligonucleotide is further synthesized. This process can also be carried out in the reverse sequence.
In a convergent process, for example, p-RNA oligomers having amino-terminal linkers and, for example, DNA oligomers are synthesized in separate processes using, for example, thiol linkers. An iodoacetylation of the p-RNA oligomer and the coupling of the two units according to protocols known from the literature is preferably then carried out (T. Zhu et al., Bioconjug. Chem. 1994, 5, 312).
Convergent processes have proved to be particularly preferred on account of their flexibility.
The term conjugate within the meaning of the present invention is also understood as meaning so-called arrays. Arrays are arrangements of immobilized recognition species which, especially in analysis and diagnosis, play an important role in the simultaneous determination of analytes. Examples are peptide arrays (Fodor et al., Nature 1993, 364, 555) and nucleic acid arrays (Southern et al. Genomics 1992, 13, 1008; Heller, U.S. Pat. No. 5,632,957). A higher flexibility of these arrays can be achieved by bonding the recognition species to coding oligonucleotides and the associated, complementary strands to certain positions on a solid carrier. By applying the coded recognition species to the xe2x80x9canti-codedxe2x80x9d solid carrier and adjustment of hybridizaton conditions, the recognition species are non-covalently bonded to the desired positions. As a result, various types of recognition species, such as, for example, DNA sections, antibodies, can be arranged simultaneously on a solid carrier only by use of hybridization conditions (see FIG. 4.). As a prerequisite for this, however, codons and anticodons are necessary which are extremely strong, selectivexe2x80x94in order to keep the coding sections as short as possiblexe2x80x94and do not interfere with natural nucleic acid. p-NAs, preferably p-RNAs, are particularly advantageously suitable for this.
Another subject of the present invention is therefore a carrier on which is immobilized at least one linker nucleoside according to the invention, at least one nucleic acid according to the invention and/or at least one conjugate according to the invention.
The term xe2x80x9cimmobilizedxe2x80x9d is understood within the meaning of the present invention as meaning the formation of a covalent bond, quasi-covalent bond or supramolecular bond by association of two or more molecular species such as molecules of linear constitution, in particular peptides, peptoids, proteins, linear oligo- or polysaccharides, nucleic acids and their analogues, or monomers such as heterocycles, in particular nitrogen heterocycles, or molecules of non-linear constitution such as branched oligo- or polysaccharides or antibodies and their functional moieties such as Fv fragments, single chain Fv fragments (scFv) or Fab fragments.
Suitable carrier materials are, for example, ceramic, metal, in particular noble metal, glasses, plastics, crystalline materials or thin layers of the carrier, in particular of the materials mentioned, or (bio)molecular filaments such as cellulose, structural proteins.
A further subject of the present invention also relates to a diagnostic comprising a linker nucleoside according to the invention, a nucleic acid according to the invention or a conjugate according to the invention, as already described in greater detail above.
Another subject of the invention is therefore the use of a linker nucleoside according to the invention, of a nucleic acid according to the invention, of a conjugate according to the invention and/or of a carrier according to the invention for the production of a pharmaceutical, such as, for example, of a therapeutic, of a diagnostic and/or of an electronic component.
The invention also relates to the use of the linker nucleosides according to the invention, of the nucleic acid according to the invention or of the conjugate according to the invention and/or of the carrier according to the invention in a pairing and/or test system, such as described in greater detail, for example, in WO94/28173, WO96/13522, WO96/13613, R. L. Letsinger, et al., Nature, 1996, 382, 607-9; P. G. Schultz et al., Nature, 1996, 382, 609-11 or A. Lombardi, J. W. Bryson, W. F. DeGrado, Biopolymers (Pept. Sci.) 1997, 40, 495-504 and generally explained above.
The following figures and examples are intended to describe the invention in greater detail, without restricting it.